This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. At Rib-X Pharmaceuticals, Inc., we employ a structure-based design strategy to find novel chemical classes of antibiotics that target the ribosome. The medical need for new drugs that circumvent established antibiotic resistance mechanisms in bacteria is dire, with many common drug classes such as penicillins and macrolides having high levels of community resistance in many parts of the world. Though many antibiotics target other aspects of the bacterial life cycle, those that bind to the translational apparatus constitute a majority of use. The central player in translation is the ribosome, a 2.5MDa macromolecular complex that uniquely catalyzes the synthesis of proteins in all organisms that has been the target of many antibiotics, both natural and synthetic. Using our constantly improving knowledge of important drug-RNA binding interactions, we are currently pursuing the design of additional next generation antibiotics based on different novel molecular scaffolds. For such, we propose to accumulate more structural information using our crystals of H. marismortui 50S bound to novel inhibitors of protein translation synthesized in house. The structural information we obtain from our H. marismortui 50S crystals will be essential for the subsequent design of compounds with increasingly improved drug-like properties within our current programs. Furthermore, the sheer size of the 50S subunit and the myriad of macro-molecular interactions inherent in the translational apparatus, affords many small molecule binding locations that would interfere with translation. A substantial number of these regions are distal to the peptidyl-transferase center and are not well conserved between the archeal and eubacterial kingdoms. Therefore, we also propose to determine the crystal structures of several inhibitors bound to the 50S ribosomal subunit from a Gram-positive bacterium. These structures will allow us to the exploit molecular differences between archeal and eubacterial ribosome, and employ our iterative structure based drug design methodology on these essential yet less well conserved sites.